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Influences of transporter associated with antigen processing (TAP) on the repertoire of peptides associated with the endoplasmic reticulum-resident stress protein gp96.

Arnold D, Wahl C, Faath S, Rammensee HG, Schild H - J. Exp. Med. (1997)

Bottom Line: This ability is based on peptides associated with gp96.We found that for some antigens the association of peptides with gp96 required functional TAP molecules, whereas the association of peptides from other antigens was TAP independent.These results, together with the earlier finding that the gp96 peptide repertoire is independent of the major histocompatibility complex molecules expressed by the cell gp96 is isolated from, give the theoretical foundation for the ability of gp96 to induce CTL responses against all kinds of intracellular antigens.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Institute of Cell Biology, Eberhard-Karls-University, Tübingen, Germany.

ABSTRACT
The endoplasmic reticulum (ER)-resident stress protein gp96 induces protective immunity and specific cytotoxic T lymphocyte (CTL) responses against antigens expressed in those cells it has been isolated from. This ability is based on peptides associated with gp96. Because gp96 is located inside the ER, our experiments address the question whether or not the repertoire of peptides associated with gp96 is influenced by the transporter associated with antigen processing (TAP). For this purpose, gp96 was isolated from cells with and without a TAP defect and used for immunization of mice. We found that for some antigens the association of peptides with gp96 required functional TAP molecules, whereas the association of peptides from other antigens was TAP independent. In the case of a TAP-dependent association of peptides with gp96, our results prove that peptide binding by gp96 in vivo occurs inside the ER and is not an artifact induced by cell lysis during the gp96 purification. The finding that some antigens can also associate with gp96 in the absence of functional TAP molecules indicates that the repertoire of peptides bound by gp96 truly reflects the entire repertoire of peptides present inside the ER and not only those peptides transported by TAP. These results, together with the earlier finding that the gp96 peptide repertoire is independent of the major histocompatibility complex molecules expressed by the cell gp96 is isolated from, give the theoretical foundation for the ability of gp96 to induce CTL responses against all kinds of intracellular antigens.

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TAP-independent recognition of minor H antigens by  BALB.B anti-C57BL/6 CTL. BALB.B mice were immunized with  C57BL/6 spleen cells (closed symbols) or left untreated (open symbols) and  stimulated in vitro with irradiated C57BL/6 cells. CTLs were tested for  their ability to recognize RMA cells (triangles) or RMA-S cells (circles).
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Figure 4: TAP-independent recognition of minor H antigens by BALB.B anti-C57BL/6 CTL. BALB.B mice were immunized with C57BL/6 spleen cells (closed symbols) or left untreated (open symbols) and stimulated in vitro with irradiated C57BL/6 cells. CTLs were tested for their ability to recognize RMA cells (triangles) or RMA-S cells (circles).

Mentions: In the following experiments, we analyzed whether the TAP-dependent peptide loading of gp96 molecules is a general phenomenon that can be observed for other antigens as well. Therefore, BALB/c mice (H-2d) were immunized with gp96 molecules from R/gal and RS/gal cells to induce cross-priming against minor H antigens expressed in C57BL/6 mice (H-2b), from which R/gal and RS/gal cells were generated. The spleen cells from the immunized mice were stimulated with B10.D2 splenocytes (H-2d), sharing most of the minor H antigens with C57BL/6. As shown in Fig. 3, spleen cells from the untreated mice showed no CTL activity, because the induction of minor H-specific CTL requires in vivo priming (15), but spleen cells from mice immunized with gp96 preparations from both R/gal and RS/gal displayed CTL activity against B10.D2 Con A blasts. A contamination of gp96 molecules from RS/gal cells with those from R/gal cells can be excluded because in some experiments spleen cells from mice immunized with RS/gal gp96 were also stimulated with β-gal peptides and never showed β-gal–specific CTL activity. The results could also be reproduced with gp96 molecules from untransfected RMA and RMA-S cells (data not shown). Therefore, these experiments show that the loading of peptides, reflecting some of the minor H antigens that differ between BALB and C57BL mice, onto gp96 molecules is independent of TAP. The origin of these minor H antigens is not known, but obviously their peptides enter the ER and are loaded onto gp96 molecules by a TAP-independent mechanism. This is not a rare phenomenon because some minor H antigens can be also recognized by H-2b–restricted CTL in a TAP-independent fashion, as shown earlier (14) and again in Fig. 4. RMA and RMA-S cells are lysed comparably by CTL specific for minor H antigens differing between BALB.B and C57BL/6 mice, now presented by MHC molecules of the H-2b haplotype. TAP-independent loading of peptides onto gp96 and MHC class I molecules is not observed in all minor H incompatible strain combinations. When we immunized 129/Sv mice with gp96 molecules from RMA or RMA-S cells to induce a CTL response against minor H-antigens that differ between 129/Sv and C57BL/6 mice, we observed that only gp96 molecules from RMA but not RMA-S cells were able to generate a minor H-specific CTL response (Fig. 5 A). This is in contrast with the experiments shown in Fig. 3 where the identical RMA-S gp96 preparation induced a CTL response in BALB/c mice against B10.D2 Con A blasts. In line with the above results, only RMA but not RMA-S cells are recognized by the minor H-specific CTL induced by immunization of 129/Sv mice with C57BL/6 cells or RMA-derived gp96 molecules (Fig. 5 B). This supports the notion that peptide loading onto MHC and gp96 molecules happens in the same intracellular compartment.


Influences of transporter associated with antigen processing (TAP) on the repertoire of peptides associated with the endoplasmic reticulum-resident stress protein gp96.

Arnold D, Wahl C, Faath S, Rammensee HG, Schild H - J. Exp. Med. (1997)

TAP-independent recognition of minor H antigens by  BALB.B anti-C57BL/6 CTL. BALB.B mice were immunized with  C57BL/6 spleen cells (closed symbols) or left untreated (open symbols) and  stimulated in vitro with irradiated C57BL/6 cells. CTLs were tested for  their ability to recognize RMA cells (triangles) or RMA-S cells (circles).
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Related In: Results  -  Collection

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Figure 4: TAP-independent recognition of minor H antigens by BALB.B anti-C57BL/6 CTL. BALB.B mice were immunized with C57BL/6 spleen cells (closed symbols) or left untreated (open symbols) and stimulated in vitro with irradiated C57BL/6 cells. CTLs were tested for their ability to recognize RMA cells (triangles) or RMA-S cells (circles).
Mentions: In the following experiments, we analyzed whether the TAP-dependent peptide loading of gp96 molecules is a general phenomenon that can be observed for other antigens as well. Therefore, BALB/c mice (H-2d) were immunized with gp96 molecules from R/gal and RS/gal cells to induce cross-priming against minor H antigens expressed in C57BL/6 mice (H-2b), from which R/gal and RS/gal cells were generated. The spleen cells from the immunized mice were stimulated with B10.D2 splenocytes (H-2d), sharing most of the minor H antigens with C57BL/6. As shown in Fig. 3, spleen cells from the untreated mice showed no CTL activity, because the induction of minor H-specific CTL requires in vivo priming (15), but spleen cells from mice immunized with gp96 preparations from both R/gal and RS/gal displayed CTL activity against B10.D2 Con A blasts. A contamination of gp96 molecules from RS/gal cells with those from R/gal cells can be excluded because in some experiments spleen cells from mice immunized with RS/gal gp96 were also stimulated with β-gal peptides and never showed β-gal–specific CTL activity. The results could also be reproduced with gp96 molecules from untransfected RMA and RMA-S cells (data not shown). Therefore, these experiments show that the loading of peptides, reflecting some of the minor H antigens that differ between BALB and C57BL mice, onto gp96 molecules is independent of TAP. The origin of these minor H antigens is not known, but obviously their peptides enter the ER and are loaded onto gp96 molecules by a TAP-independent mechanism. This is not a rare phenomenon because some minor H antigens can be also recognized by H-2b–restricted CTL in a TAP-independent fashion, as shown earlier (14) and again in Fig. 4. RMA and RMA-S cells are lysed comparably by CTL specific for minor H antigens differing between BALB.B and C57BL/6 mice, now presented by MHC molecules of the H-2b haplotype. TAP-independent loading of peptides onto gp96 and MHC class I molecules is not observed in all minor H incompatible strain combinations. When we immunized 129/Sv mice with gp96 molecules from RMA or RMA-S cells to induce a CTL response against minor H-antigens that differ between 129/Sv and C57BL/6 mice, we observed that only gp96 molecules from RMA but not RMA-S cells were able to generate a minor H-specific CTL response (Fig. 5 A). This is in contrast with the experiments shown in Fig. 3 where the identical RMA-S gp96 preparation induced a CTL response in BALB/c mice against B10.D2 Con A blasts. In line with the above results, only RMA but not RMA-S cells are recognized by the minor H-specific CTL induced by immunization of 129/Sv mice with C57BL/6 cells or RMA-derived gp96 molecules (Fig. 5 B). This supports the notion that peptide loading onto MHC and gp96 molecules happens in the same intracellular compartment.

Bottom Line: This ability is based on peptides associated with gp96.We found that for some antigens the association of peptides with gp96 required functional TAP molecules, whereas the association of peptides from other antigens was TAP independent.These results, together with the earlier finding that the gp96 peptide repertoire is independent of the major histocompatibility complex molecules expressed by the cell gp96 is isolated from, give the theoretical foundation for the ability of gp96 to induce CTL responses against all kinds of intracellular antigens.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Institute of Cell Biology, Eberhard-Karls-University, Tübingen, Germany.

ABSTRACT
The endoplasmic reticulum (ER)-resident stress protein gp96 induces protective immunity and specific cytotoxic T lymphocyte (CTL) responses against antigens expressed in those cells it has been isolated from. This ability is based on peptides associated with gp96. Because gp96 is located inside the ER, our experiments address the question whether or not the repertoire of peptides associated with gp96 is influenced by the transporter associated with antigen processing (TAP). For this purpose, gp96 was isolated from cells with and without a TAP defect and used for immunization of mice. We found that for some antigens the association of peptides with gp96 required functional TAP molecules, whereas the association of peptides from other antigens was TAP independent. In the case of a TAP-dependent association of peptides with gp96, our results prove that peptide binding by gp96 in vivo occurs inside the ER and is not an artifact induced by cell lysis during the gp96 purification. The finding that some antigens can also associate with gp96 in the absence of functional TAP molecules indicates that the repertoire of peptides bound by gp96 truly reflects the entire repertoire of peptides present inside the ER and not only those peptides transported by TAP. These results, together with the earlier finding that the gp96 peptide repertoire is independent of the major histocompatibility complex molecules expressed by the cell gp96 is isolated from, give the theoretical foundation for the ability of gp96 to induce CTL responses against all kinds of intracellular antigens.

Show MeSH
Related in: MedlinePlus